This invention relates to the Magnetic Resonance Imaging (M.R.I.), a technique used in the medical diagnosis field for a number of years, to rapidly detect a series of anomalies and/or pathological conditions of living human or animal body organs or tissues. (i. e.: Stark D. D., Bradley W. G. Jr., Eds.: xe2x80x9cMagnetic Resonance Imagingxe2x80x9d, the C.V. Mosby Company, St. Louis, Mo. (USA), 1988). In particular, the invention relates to new chelating agents, especially aminopolycarboxylic acid derivative compounds and to metal chelates thereof with bivalent or trivalent paramagnetic ions and/or salts thereof as well as their use as M.R.I. contrast agents.
Diagnostic imaging techniques, such as Magnetic Resonance Imaging have been used in medical diagnosis for a long time. The use of contrast media to improve tissue differentiation, to delineate structures or monitor physiological functions constitutes in some cases a fundamental contribution in the best formulation of some medical diagnosis and a valid support for radiologist work.
The medical use of aminopolycarboxylic acid or carboxylic acid derivatives and metal chelates thereof as M.R.I. contrast agents is well known. Said contrast agents, to simplify, can be seen as pertaining to two main groups: the linear and the cyclic ones.
The present invention relates to linear polyaminopolycarboxylic acid derivatives, as well as their complexes with paramagnetic metal ions, in particular the Gd3+ ion.
Patent literature is rich in patent and patent applications relating to the use of linear polyaminopolycarboxylic acid derivatives in the preparation of MRI contrast agents. These compounds generally are derived from the simplest one, N,N,Nxe2x80x2,Nxe2x80x3,Nxe2x80x3-diethylenetriamine-pentaacetic acid, (DTPA), of which the Meglumine salt of the Gd3+ complex has been commercialised for a number of years as MAGNEVIST(trademark). To improve stability, water solubility and selectivity and to reduce toxicity of these contrast agents generally patent literature proposes the preparation of esters or amide derivatives of said acids or the introduction of substituents on the diethylene unit of the diethylenetriamine DTPA skeleton. As an example of said patent literature we can cite: Guerbet EP 661279; Concat Ltd., WO 95/05118; Dibra WO 95/15319; Mallinckrodt WO 94/08630; Green Gross Corp. JP 06016606 and JP 05229998; Mallinckrodt U.S. Pat. Nos. 5,141,740 and 5,077,037; Cockbain-Nycomed WO 91/15467 and WO 92/11232; Salutar U.S. Pat. Nos. 4,889,931 and 4,858,451; Abbot Laboratoires EP 279307; Nycomed EP 299795; Metasyn Inc. WO 95/28179; Schering EP 680 464; and document cited in these patent publications. Some documents further exist in which substituents have been introduced in (xcex1) to one or more carboxylic DTPA groups; for example: Bracco EP-B-230893 and U.S. Pat. No. 5,182,370; Schering WO 96/16928, WO 96/16929, WO 96/26180 and DE 4341724 enclosing xcex1 derivatives, generally comprising an aromatic group, particularly useful for the imaging of the hepatobiliary system. In particular, some patent literature further exists, in which the introduction of an aromatic or lipophilic group on the chelant structure is specifically stated to make the contrast agent as particularly useful for a best definition of the liver and the biliary duct: the General Hospital Corporation U.S. Pat. No. 4,899,755 and WO-A-86/06605.
Further relevant documents disclosing DTPA derivatives which carry substituents in xcex1 position to the carboxy groups of the DTPA skeleton exist. Among the others, U.S. Pat. No. 5,885,548 describes DTPA analogs carrying a radicals which comprise at least one aryl group. No alicyclic substituents are indicated as possible substituents. The chelate complex of the invention are strongly preferred for the imaging of the liver, the bile ducts and the gallbladder, wichever the diagnostic technique is. U.S. Pat. No. 5,853,699 describes a range of compounds similar to the preceiding ones. In this case the preferred and strongly recommended ones are tetraazamacrocycles and are described and claimed only as contrast agents for the imaging of the liver by using the Computer Tomography, i. e. X-rays. No mention of a possible use with the Magnetic Resonance is done nor even suggested. DTPA derivatives xcex1 substituted, whereas the possible xcex1 substituents are only lower alkyl groups, and where said derivatives are in any case mono or diamido compounds are also disclosed in U.S. Pat. No. 5,453,264. No mention of possible cyclic moieties in the xcex1 substituents is done or suggested. Also U.S. Pat. No. 5,672,335 discloses DTPA derivatives with possible substituents, comprising at least one aromatic ring, in xcex1 position to the carboxy groups. Even this patent only suggests the imaging of the liver and bile ducts by using the X-rays (Computer Tomography), not magnetic resonance. The general teaching of the prior-art concerns the possibility of finding compounds that are particularly useful for the imaging of liver, bile ducts, gall bladder provided that lipophilic substituents are attached to the chelating moiety. More lipophilic substituents are highly preferred/needed for obtaining liver specificity. No mention, nor even suggestion is made to the possibility that such DTPA derivatives can show very high relaxivity values in serum, and, on consequence, the capability of strongly bonding to the serum proteins.
The compounds of the present invention are diethylenetriaminepentaacetic acid derivatives characterised in having substituents at the a position to the carboxy group of two or three of the five acetic groups of DTPA. More precisely, the compounds can have two substituents (the same or different from each other) in xcex1 to the carboxyls of the two acetic groups respectively bound to the two side nitrogen atoms of DTPA; or they can have three substituents (the same or different from each other) in xcex1 to the carboxyl groups of three acetic groups respectively bound to the three nitrogen atoms of DTPA.
Therefore, the compounds of the present invention are characterized in having some sterical hindrance, due to the presence of two or three substituents at the above mentioned positions. The minimum size of the substituents is that of a chain having at least three carbon atoms.
Said hindering groups are probably responsible for the interactions of the paramagnetic chelates with biological components of the fluids in which the agent diffuses, said interactions substantially consisting in the formation of strong non-covalent bonds with the proteins present in such fluids. In particular said tight bonding is detectable in plasma, showing a great affinity of the compounds of the invention with the plasma proteins. This feature is particularly unexpected in view of the general teaching of the art. Actually it is well known that when a compound is hepatospecific, it is preferentially/selectively taken up by the liver hepatocytes and then it is excreted via the biliary route, in such way being cleared from the palsma. The compounds of the present invention, on the contrary, showed such an affinity with the plasma proteins to give extremely high values of relaxivity in serum (thus confirming the formation of strong bonds with serum proteins). Accordingly one of their preferential and strongly recommended use is as potential blood pool contrast agents, said caracteristic feature giving the possibility of a long period of permanence inside the vessels even administering substantially lower doses.
Relaxivity values of the contrast agent of the present invention have been tested either in saline or in human serum obtained by Seronorm(trademark) Human, freeze-dried human serum produced by Nycomed Pharma AS, Oslo, Norway. Serum obtained from said Seronorm(trademark) is substantially equivalent to the fresh one, so its use in the relaxivity determination grants a good picture of the xe2x80x9cin vivoxe2x80x9d behaviour and, further, an excellent reproducibility of this test.
The compounds object of the present invention are characterised by very high r1 and r2 relaxivity values. When measured in Seronorm(trademark) Human at 20 MHz, at a temperature of 39xc2x0 C., and at a concentration comprised from 0 to 1 mM, the compounds of the present invention have r1 relaxivity equal to or, preferably, higher than 15 sxe2x88x921mmxe2x88x921.
The present invention relates to novel chelating agents, more particularly linear aminopolycarboxylic acid derivatives chelants, and metal chelates thereof and the use of such chelating agents and chelates in the preparation of diagnostic imaging contrast agents and in particular of contrast agents exhibiting improved serum relaxivity.
Said compounds are polyaminopolycarboxylic acid derivatives of formula (I), either in their racemic or enantiomeric forms: 
wherein:
R is H or a linear or branched, saturated or unsaturated C1-C20 alkyl chain, which is interrupted or not by one or more O, N, S atoms or by one or more xe2x80x94COxe2x80x94, xe2x80x94CH(OH)xe2x80x94, xe2x80x94CH(NH2)xe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NHxe2x80x94, which is substituted or not with one or more halogen atoms or xe2x80x94COOH groups or their ester or amide derivatives and which is interrupted or not or substituted or not by one or more cyclic R3 residues which can be the same or different and isolated or fused, with the proviso that, if some of said residues are fused, the maximum number of rings forming the corresponding polycyclic unit is three, in which:
R3 is a 5- or 6-membered carbocyclic or heterocyclic, saturated, unsaturated or aromatic cyclic unit, substituted or not with one or more groups X, which can be the same or different, in which:
X is OH, halogen, NH2, NHL, N(L)2, xe2x80x94Oxe2x80x94L, xe2x80x94Sxe2x80x94L, xe2x80x94COxe2x80x94L, where L, the same or different from each other, is C1-C5 linear or branched alkyl, substituted or not with one or more hydroxy, alkoxy or carboxylic groups, or X is a COOH group or its ester or amide derivative, or a xe2x80x94SO3H group or its amide derivative, and
R1, R2 have the same meanings as R, independently from each other, except H, with the proviso that: when R1 and R2 are both C6H5xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94, R is different from either H or C6H5xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94.
The invention further relates to complexes of the ligand of formula (I) with metal ions of atomic number from 20 to 31, 39, from 42 to 44, 49 and from 57 to 83; particularly preferred metals being: Fe(2+), Fe(3+), Cu(2+), Cr(3+), Gd(3+), Eu(3+), Dy(3+), La(3+), Yb(3+), Mn(2+); as well as, where the metal chelate carries an overall charge, a salts thereof with a physiologically acceptable counterion, preferably selected from organic bases such as a primary, secondary or tertiary amines, a basic amino acid, or an inorganic base derived from an alkali metal or alkaline-earth metal cation such as: Na+, K+, Mg2+, Ca2+ or a mixture thereof.
The present invention further relates to the use of the compounds of formula (I) and of the salts of the complexes thereof as well as to the pharmaceutical formulations containing them for a diagnostic or therapeutic scope.
Preferred are the compounds of formula (I) in which R, R1 and R2 are selected from the following groups: 
Among the compounds formula (I), particularly preferred are the compounds of formula (II): 
where:
R4=H, or a linear or branched C1-C10 alkyl, optionally interrupted by one or more xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94COxe2x80x94 groups and/or N, O, S atoms, optionally interrupted or substituted with 1 to 3 saturated rings, that are optionally interrupted by one or more N, O, S and that are optionally substituted with xe2x80x94OH, xe2x80x94SH, halogen, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2, xe2x80x94CON(Rxe2x80x3)2, xe2x80x94SO3H, C1-C4 alkoxy groups;
R5=independently a linear or branched C1-C10 alkyl, optionally interrupted by one or more xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94COxe2x80x94groups and/or N, O, S atoms and interrupted or substituted with 1 to 3 saturated rings, that are optionally interrupted by one or more N, O, S and that are optionally substituted with xe2x80x94OH, xe2x80x94SH, halogen, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2, xe2x80x94CON(Rxe2x80x3)2, xe2x80x94SO3H, C1-C4 alkoxy groups;
Rxe2x80x3=independently H or C1-C5 linear or branched alkyl, optionally substituted with from 1 to 5 xe2x80x94OH groups.
Equally preferred are the compounds of formula (III): 
where:
R6=H, or a linear or branched C1--C10 alkyl, optionally interrupted by one or more xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94COxe2x80x94 groups and/or N, S atoms and optionally substituted with one or more xe2x80x94OH, xe2x80x94NH2, xe2x80x94COOH groups;
R7=independently a linear or branched C2-C10 alkyl, optionally interrupted by one or more xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94COxe2x80x94 groups and/or N, S atoms and optionally substituted with one or more xe2x80x94OH, xe2x80x94NH2, xe2x80x94COOH groups.
Equally preferred are the compounds of formula (IV): 
where:
R8=H, or a linear or branched C1-C10 alkyl, optionally interrupted by one or more xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94COxe2x80x94 groups and/or N, S atoms, optionally interrupted or substituted with 1 to 3 isolated or fused saturated, unsaturated or aromatic rings, that are optionally interrupted by one or more N, O, S and that are optionally substituted with one or more xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2 C1-C6 alkyl, C1-C6 alkoxy, C6-C20 arylalkoxy groups;
R9=independently a linear or branched C1-C6 alkyl, optionally interrupted by one or more xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94COxe2x80x94 groups and/or N, S atoms, which is interrupted or substituted with 2 to 3 fused saturated, unsaturated or aromatic rings, that are optionally interrupted by one or more N, O, S and that are optionally substituted with one or more xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2, C1-C6 alkyl, C1-C6 alkoxy, C6-C20 arylalkoxy groups;
Rxe2x80x3=independently H or C1-C5 linear or branched alkyl, optionally substituted with 1 to 5 xe2x80x94OH groups.
Equally preferred are the compounds of formula (V): 
where:
R10=a linear or branched C1-C10 alkyl, optionally interrupted by one or more xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94COxe2x80x94 groups and/or N, S atoms, interrupted or substituted with 1 to 3 saturated, unsaturated or aromatic rings, that are optionally interrupted by one or more N, O, S and that are optionally substituted with one or more xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2, C1-C6 alkyl, C1-C6 alkoxy groups;
R11=independently a linear or branched C2-C10 alkyl, optionally interrupted by one or more N, S atoms.
Two further groups of preferred compounds, all included in compounds of formula (I), are the compounds of formula (VI) 
where:
R12=a linear or branched C2-C10 alkyl, optionally interrupted by one or more xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94COxe2x80x94 groups and/or N, S atoms, optionally substituted with one or more xe2x80x94COOH, xe2x80x94NH2 groups, optionally interrupted or substituted with 1 to 3 saturated, unsaturated or aromatic, isolated or fused rings, that are optionally interrupted by one or more N, O, S and that are optionally substituted with one or more xe2x80x94OH, xe2x80x94COOH, xe2x80x94NH2, xe2x80x94N(Rxe2x80x3)2, C1-C6 alkyl, C1-C6 alkoxy groups,
and the compounds of formula(VII) 
where:
R13=H, linear or branched C1-C6 alkyl, substituted or interrupted with 1 aromatic ring, that is optionally interrupted by one or more N, O, S;
R14=independently linear or branched C1-C6 alkyl, substituted or interrupted with one aromatic ring, that is optionally interrupted by one or more N, O, S.
Also preferred among the compounds of the formula (I), are the compounds of formula (VIII): 
where:
R15=independently H, halogen;
R16=H, OH, N(Rxe2x80x3)2, COORxe2x80x3, xe2x80x94CON(Rxe2x80x3)2, xe2x80x94SO3H, xe2x80x94SO2NHRxe2x80x3, C1-C6 alkyl, C1-C6 alkoxy;
R17=independently C1-C6 alkyl, substituted with xe2x80x94COOH or xe2x80x94CON(Rxe2x80x3)2 or from 1 to 3 xe2x80x94OH groups;
A=direct bond (i.e. no intervening atom), xe2x80x94Oxe2x80x94, Cxe2x95x90O
m=integer 1-6;
n=integer 0-2;
Rxe2x80x3=independently H or C1-C5 linear or branched alkyl, optionally substituted with 1 to 5 xe2x80x94OH groups
with the proviso that, when R16=H, at least one of the substituents R15 is different from hydrogen.
Particularly preferred, among the various possible synthetic pathways yielding the compounds of the invention, is the following one, which is reported in the following Scheme 1 in order to further clarify the process: 
wherein Pg=protective group (such as t-butyl);
R1 as defined for compounds of general formula (I).
Step (a) involves the protection of the alcohol group of 2-bromoethanol with dihydropyran to give intermediate (1). The reaction is carried out in an organic solvent such as CH2Cl2, CHC13, CH2ClCH2CL, in the presence of 4-toluenesulfonic acid pyridinium salt or of other acid catalysts. In intermediate (1) the Br atom can be replaced with any other nucleofugal group (such as Cl, I, xe2x80x94OMs, xe2x80x94OTf, xe2x80x94OTs) and the alcohol-protecting group can be replaced, for example, by benzyl and trityl.
In step (b) the ester (for example the t-butyl ester) of a natural or synthetic xcex1-amino acid (2), in the racemic or optically active form, is reacted with intermediate (1) in the presence of diisopropylethylamine in a solvent such as CH3CN, DMF or a chlorinated solvent, to give intermediate (3).
The latter is reacted, in step (c), with a bromoacetic acid ester (such as t-butylbromoacetate) in the presence of diisopropylethylamine, to give intermediate (4), which is reacted, in the subsequent step (d), with 4-toluenesulfonic acid pyridinium salt, or other acid catalysts, in a water/ethanol mixture, at a temperature of 20-60xc2x0 C., to give intermediate (5).
In step (e), intermediate (5) is brominated with N-bromosuccinimide in the presence of triphenylphosphine, to give compound (6).
With a similar procedure, compound (7) of formula 
is prepared, wherein R2 is as already defined for compounds of general formula (I).
The Br atom in intermediates (6) and (7) can be replaced with any other nucleofugal group (such as Cl, I, xe2x80x94OMs, xe2x80x94OTf, xe2x80x94OTs).
Intermediates (6) and (7) are then reacted, according to the following Scheme 2, to give the compounds of general formula (I). 
wherein R, R1 , R2 are as already defined for compounds of general formula (I).
Step (f) involves the alkylation of the ester of a natural or synthetic xcex1-amino acid (8) with bromoethyl-derivative (6) using double phase conditions in acetonitrile/aqueous phosphate buffer at pH 8 in a 1:1 molar ratio between the two reagents to give compound (9).
Intermediate (9) is further alkylated with the bromoethyl derivative (7), in step (g), in the same conditions, to give the intermediate pentaester (10), which is deprotected, in step (h), in conventional conditions, to give the corresponding pentaacid. When R1 and R2 are the same, the dialkylation product (10) can be obtained directly operating in acetonitrile/aqueous phosphate buffer at pH 8 and in a aminoester (8) to bromoderivative (6) molar ratio ranging from 1:2 to 1:3.
An alternative procedure for the preparation of intermediate (6), and similarly of intermediate (7), is illustrated in the following Scheme 3: 
wherein:
Pg=protective group (such as t-butyl);
R1 is above defined for compounds of general formula (I).
Step (axe2x80x2) involves the condensation of the ester of a natural or synthetic xcex1-amino acid (2) with an xcex1-haloacetic acid ester (2) (such as 2-bromoacetic acid t-butyl ester) in double phase conditions in acetonitrile/aqueous phosphate buffer at pH 8, to. give the iminodiacetic acid derivative (2xe2x80x2), which is alkylated, in step (bxe2x80x2) in 1,2-dibromoethane as the solvent, under reflux, in the presence of N,N-diisopropylethylamine and at a temperature of about 80xc2x0 C., to give intermediate (6).
Table 1 above discloses the high relaxivity shown in serum by the compounds of the present invention; r1 and r2 relaxivity values of some of the preferred compounds are reported, in comparison with the corresponding r1 and r2 values measured for some of the mayor prior-art compounds: Gd-DTPA Dimeglumine salt (MAGNEVIST(copyright)); Gd-BOPTA Dimeglumine salt and Gd-EOB-DTPA Dimeglumine salt.
The data of Table 1 clearly show that the compounds of the present invention have surprisingly high relaxivity values r1 and r2, measured in Seronorm(trademark) Human, thus confirming the formation of unexpectedly strong non-covalent bonds with serum proteins. The ralaxivity values measured in serum for the other compounds of the invention, in particular for compounds 13 and 14 of Examples 18 and 19, are of the same order of magnitude as the ones reported in Table 1.
This is particularly interesting from the application point of view, both as far as the improvement in the obtainable images, the development of formulations specific to blood pool use and the determination of optimum low dosages of the contrast medium are concerned.